Infrastructure development often leads to considerable changes in the land use. These changes are major causes of habitat fragmentation and ecosystem loss. Moreover, decrease of the environmental impacts on biodiversity is among the most important objectives of sustainable development. For this purpose, Environmental Impact Assessment (EIA) along with the other appropriate tools can be applied to identify and predict the magnitude of such problems. Biodiversity Impact Assessment (BIA) as a specific disciplinary tool could be useful to identify the actual impacts on biodiversity within Environmental Impact Assessment. This tool with the assistance of Geographic Information system (GIS) techniques evaluates the data in a comprehensible way. In this paper, a brief case study dealing with the assessment of road alternatives has been carried out to demonstrate the efficiency of BIA. It is found that according to vegetation and wildlife maps, ecosystem loss and fragmentation score of proposed road are more than the existing one. On the basis of the assessment results, the authors also stated that application of BIA in Iran with an exclusive biodiversity is essentially needed.